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Abstract:

Embodiments of the invention relate generally to ferromagnetic
microdisks, methods of detecting target bioanalyte using ferromagnetic
microdisks, and kits (such as for using in the laboratory setting)
containing the reagents necessary to make, and/or use ferromagnetic
microdisks for bioanalyte detection, depending on the user's planned
application. The methods and products allow the fabrication of
ferromagnetic microdisks, and their use in the detection of biological
molecules with high sensitivity, little or no signal decay, improved
safety, convenience, and lowered cost for use and disposal.

Claims:

1-17. (canceled)

18. A method comprising: attaching one or more molecular probes to a
ferromagnetic microdisk to, wherein the ferromagnetic microdisk comprises
a ferromagnetic material and a unique resonance frequency; contacting the
molecular probe to at least one target bioanalyte; binding the molecular
probe to the target bioanalyte; and detecting the unique resonance
frequency of the ferromagnetic microdisk, thereby detecting the
ferromagnetic microdisk and the presence of the target bioanalyte.

19. The method of claim 18, wherein the molecular probe is selected from
the group consisting of DNA, RNA, antibody, and protein.

21. The method of claim 18 wherein the target bioanalyte is present in a
solid sample.

22. The method of claim 18 wherein the target bioanalyte is present in a
solution.

23. The method of claim 18 wherein the target bioanalyte is bound to a
solid or semisolid support.

24. The method of claim 18 wherein the ferromagnetic disk or molecular
probe is bound to a solid or semisolid support.

25. The method of claim 18 wherein the target bioanalyte is present in
vivo.

26. The method of claim 18 wherein the target bioanalyte is present in
vitro.

27. The method of claim 18 wherein the target bioanalyte is present in a
non-living sample.

28-43. (canceled)

Description:

FIELD OF INVENTION

[0001] Embodiments of the invention relate to ferromagnetic microdisks
bioconjugated to molecular probes, and methods of using bioconjugated
ferromagnetic microdisks for detecting biological molecules
(bioanalytes).

BACKGROUND

[0002] The ability to detect and identify trace quantities of analytes has
become increasingly important in virtually every scientific discipline,
ranging from part per billion analyses of pollutants in sub-surface water
to analysis of cancer treatment drugs in blood serum.

[0003] With the advancement of technologies to make and detect
biomolecules, there are multiple techniques that promise biological
detection with single molecule sensitivity. However, many of these
techniques have not yet found commercial applications or feasibility. The
main reasons are the complexity associated with these ultra-sensitive
methods, the costs, and the potential biohazards associated with the
reagents. Many methods require multiple steps of chemical treatments,
bulky and expensive instruments, and/or extreme care in sample handling
and observation. These are not ideal for practical applications that
require easy and reliable measurements that are flexible enough for
user's needs.

[0004] Additionally, many of the currently used methods of detecting
bioanalytes rely on markers or "tags" that bind to the bioanalytes and
are detected, thereby indirectly detecting the bioanalytes(s) of
interest. However, the markers or tags such as radioisotope-labeled
probes, or fluorescent markers, can lose their signal intensity over
time. For example, radioisotopes commonly used as "tags" or "markers"
decay over time, causing a gradual loss of signal that can be detected.
Because of this, some experiments need to be conducted rapidly before the
signal decays beyond the limits of detection. Similarly, fluorescent
probes are subject to "photobleaching" wherein exposure to ambient light
causes the fluorescent probe to bleach or fade away. Again, often
experiments need to be conducted quickly before photobleaching occurs, or
inconveniently in a dark setting so as to avoid photobleaching.

[0005] Safety is another consideration. Radioactive labels and their
required reagents must be used in carefully monitored situations due to
their known biologic hazards. Radioactive wastes produced from common
detection methods must be carefully disposed of so as to avoid
environmental contamination. Similarly, the toxicity of cadmium in
quantum dots and relatively large size of dye-loaded particles have
limited their applications. Although very small size (down to 10 nm in
diameter) detection has been achieved for conjugated polymer particles,
their signal intensity is lower than the larger fluorescent particles.
Lower signal intensity makes the particles more difficult to detect with
conventional techniques.

[0006] Finally, the cost of radioactive and fluorescent substances can be
substantial, both in terms of acquisition, use, safety monitoring, and
their proper monitoring and disposal.

[0007] Accordingly, there is a need for a reagents methods of bioanalyte
detection wherein the marker to be detected exhibits little or no signal
decay, and can be safely utilized in a variety of environments without
posing risks to the user or to the environment. Preferably, a marker
would have a high safety profile, exhibit a long (non or low-decaying)
signal intensity, and be available to users at a low cost for reagent use
and disposal.

[0011] Embodiments of the invention relate to ferromagnetic microdisks
bioconjugated to molecular probes, and methods of using bioconjugated
ferromagnetic microdisks for detecting biological molecules (bioanalytes)
with high sensitivity and improved ease of use and safety profiles. The
embodiments are especially directed to making and utilizing conjugated
ferromagnetic microdisks that exhibit a unique resonance frequency
depending on the geometry of the ferromagnetic microdisk, in which the
resonance frequency can be detected by appropriate instruments for the
detection of one or more bioanalyte of interest. Because the resonance
frequency exhibited by the ferromagnetic microdisk is a magnetic signal,
it does not decay or diminish over time. The invention transcends several
scientific disciplines such as polymer chemistry, biochemistry, molecular
biology, medicine, and medical diagnostics.

[0012] As used in the specification and claims, the singular forms "a,"
"an," and "the" include plural references unless the context clearly
dictates otherwise. For example, the term "an array" may include a
plurality of arrays unless the context clearly dictates otherwise.

[0013] A "ferromagnetic microdisk" is one or more of an intentionally
created devices that can be prepared by a variety of methods known in the
art, such as photolithography. The ferromagnetic microdisks exhibit
(i.e., emit) a unique magnetic vortex resonance depending on the geometry
of the microdisk, such as the diameter and thickness of the microdisk.
Ferromagnetic microdisks can be attached (bioconjugated) to chosen
molecular probes that are specific to various bioanalytes of interest.
The unique vortex resonance exhibited by the ferromagnetic microdisk can
be detected using common apparatus in the art.

[0014] The terms "nanomaterial" and "nanoparticle" as used herein refer to
a structure, a device, or a system having a dimension at the atomic,
molecular or macromolecular levels, in the length scale of approximately
1-1000 nanometer range, preferably in the range of about 2 m to about 200
mm, more preferably in the range of about 2 nm to about 50 nm.

[0016] The sample such as a bioanalyte in the embodiments of this
invention can be in the form of solid, liquid or gas, or solution. The
sample can be analyzed by the embodiments of the methods and devices of
this invention when the sample is at room temperature, and at lower than
or higher than the room temperature. Samples may be obtained from any
source, biologic or non-biologic.

[0017] Further, the bioanalyte could be an organic or inorganic molecule.
Some examples of analytes may include a small molecule, a biomolecule, or
a nanomaterial such as but not necessarily limited to a small molecule
that is biologically active, nucleic acids and their sequences, peptides
and polypeptides, as well as nanostructure materials chemically modified
with biomolecules or small molecules capable of binding to molecular
probes such as chemically modified carbon nanotubes, carbon nanotube
bundles, nanowires, nanoclusters or nanoparticles. The bioanalyte
molecule may be a fluorescently labeled molecule, such as DNA or RNA.

[0018] The term "fluid" used herein means an aggregate of matter that has
the tendency to assume the shape of its container, for example a liquid
or gas. Analytes in fluid form can include fluid suspensions and
solutions of solid particle analytes.

[0019] The term "bi-functional linker group" refers to an organic chemical
compound that has at least two chemical groups or moieties, such as for
example, carboxyl group, amine group, thiol group, aldehyde group, epoxy
group, that can be covalently modified specifically; the distance between
these groups is equal to or greater than 5-carbon bonds.

[0020] The term "molecular probe," "biomolecular probe," "capture
molecule," or "affinity agent" refers to a molecule or group/collection
of molecules that is attached ("bioconjugated"), reversibly or
irreversibly, to a ferromagnetic microdisk. The molecular probe
generally, but not necessarily, also binds to one or more bioanalytes of
interest, as described above. The biomolecular probe is typically a
nucleotide, an oligonucleotide, or a protein, but can also be a small
molecule, biomolecule, or nanomaterial such as, but not necessarily
limited to, a small molecule that is biologically active, nucleic acids
and their sequences, peptides and polypeptides, as well as nanostructure
materials chemically modified with biomolecules or small molecules
capable of binding to a target molecule that is bound to a probe molecule
to form a complex of the capture molecule, target molecule and the probe
molecule. The capture molecule may be fluorescently labeled DNA or RNA.
The capture molecule may or may not be capable of binding to just the
target bioanalyte or just the probe molecule. Other molecular probes
include, for example, antibodies, antibody fragments, antigens, epitopes,
lectins, proteins, polypeptides, receptor proteins, ligands, hormones,
vitamins, metabolites, substrates, inhibitors, cofactors,
pharmaceuticals, aptamers, cytokines and neurotransmitters.

[0021] The term "molecule" generally refers to a macromolecule or polymer
as described herein. However, SEF nanoparticles comprising single
molecules, as opposed to macromolecules or polymers, are also within the
scope of the embodiments of the invention.

[0022] A "macromolecule" or "polymer" comprises two or more monomers
covalently joined. The monomers may be joined one at a time or in strings
of multiple monomers, ordinarily known as "oligomers." Thus, for example,
one monomer and a string of five monomers may be joined to form a
macromolecule (polymer) of six monomers. Similarly, a string of fifty
monomers may be joined with a string of hundred monomers to form a
macromolecule or polymer of one hundred and fifty monomers. The term
polymer as used herein includes, for example, both linear and cyclic
polymers of nucleic acids, polynucleotides, polynucleotides,
polysaccharides, oligosaccharides, proteins, polypeptides, peptides,
phospholipids and peptide nucleic acids (PNAs). The peptides include
those peptides having either α-, β-, or ω-amino acids.
In addition, polymers include heteropolymers in which a known drug is
covalently bound to any of the above, polyurethanes, polyesters,
polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene
sulfides, polysiloxanes, polyimides, polyacetates, or other polymers
which will be apparent upon review of this disclosure.

[0023] The term "nucleotide" includes deoxynucleotides and analogs
thereof. These analogs are those molecules having some structural
features in common with a naturally occurring nucleotide such that when
incorporated into a polynucleotide sequence, they allow hybridization
with a complementary polynucleotide in solution. Typically, these analogs
are derived from naturally occurring nucleotides by replacing and/or
modifying the base, the ribose or the phosphodiester moiety. The changes
can be tailor-made to stabilize or destabilize hybrid formation, or to
enhance the specificity of hybridization with a complementary
polynucleotide sequence as desired, or to enhance stability of the
polynucleotide.

[0024] The term "polynucleotide" or "nucleic acid" as used herein refers
to a polymeric form of nucleotides of any length, either ribonucleotides
or deoxyribonucleotides, that comprise purine and pyrimidine bases, or
other natural, chemically or biochemically modified, non-natural, or
derivatized nucleotide bases. Polynucleotides of the embodiments of the
invention include sequences of deoxyribopolynucleotide (DNA),
ribopolynucleotide (RNA), or DNA copies of ribopolynucleotide (cDNA)
which may be isolated from natural sources, recombinantly produced, or
artificially synthesized. A further example of a polynucleotide of the
embodiments of the invention may be polyamide polynucleotide (PNA). The
polynucleotides and nucleic acids may exist as single-stranded or
double-stranded. The backbone of the polynucleotide can comprise sugars
and phosphate groups, as may typically be found in RNA or DNA, or
modified or substituted sugar or phosphate groups. A polynucleotide may
comprise modified nucleotides, such as methylated nucleotides and
nucleotide analogs. The sequence of nucleotides may be interrupted by
non-nucleotide components. The polymers made of nucleotides such as
nucleic acids, polynucleotides and polynucleotides may also be referred
to herein as "nucleotide polymers.

[0025] An "oligonucleotide" is a polynucleotide having 2 to 20
nucleotides. Analogs also include protected and/or modified monomers as
are conventionally used in polynucleotide synthesis. As one of skill in
the art is well aware, polynucleotide synthesis uses a variety of
base-protected nucleoside derivatives in which one or more of the
nitrogens of the purine and pyrimidine moiety are protected by groups
such as dimethoxytrityl, benzyl, tert-butyl, isobutyl and the like.

[0026] For instance, structural groups are optionally added to the ribose
or base of a nucleoside for incorporation into a polynucleotide, such as
a methyl, propyl or allyl group at the 2'-O position on the ribose, or a
fluoro group which substitutes for the 2'-O group, or a bromo group on
the ribonucleoside base. 2'-O-methyloligoribonucleotides (2'-O-MeORNs)
have a higher affinity for complementary polynucleotides (especially RNA)
than their unmodified counterparts. Alternatively, deazapurines and
deazapyrimidines in which one or more N atoms of the purine or pyrimidine
heterocyclic ring are replaced by C atoms can also be used.

[0027] The phosphodiester linkage, or "sugar-phosphate backbone" of the
polynucleotide can also be substituted or modified, for instance with
methyl phosphonates, O-methyl phosphates or phosphororthioates. Another
example of a polynucleotide comprising such modified linkages for
purposes of this disclosure includes "peptide polynucleotides" in which a
polyamide backbone is attached to polynucleotide bases, or modified
polynucleotide bases. Peptide polynucleotides which comprise a polyamide
backbone and the bases found in naturally occurring nucleotides are
commercially available.

[0028] When the macromolecule of interest is a peptide, the amino acids
can be any amino acids, including α, β, or ω-amino
acids. When the amino acids are α-amino acids, either the L-optical
isomer or the D-optical isomer may be used. Additionally, unnatural amino
acids, for example, β-alanine, phenylglycine and homoarginine are
also contemplated by the embodiments of the invention. These amino acids
are well-known in the art.

[0029] A "peptide" is a polymer in which the monomers are amino acids and
which are joined together through amide bonds and alternatively referred
to as a polypeptide. In the context of this specification it should be
appreciated that the amino acids may be the L-optical isomer or the
D-optical isomer. Peptides are two or more amino acid monomers long, and
often more than 20 amino acid monomers long.

[0030] A "protein" is a long polymer of amino acids linked via peptide
bonds and which may be composed of two or more polypeptide chains. More
specifically, the term "protein" refers to a molecule composed of one or
more chains of amino acids in a specific order; for example, the order as
determined by the base sequence of nucleotides in the gene coding for the
protein. Proteins are essential for the structure, function, and
regulation of the body's cells, tissues, and organs, and each protein has
unique functions. Examples of proteins include hormones, enzymes, and
antibodies.

[0031] The term "sequence" refers to the particular ordering of monomers
within a macromolecule and it may be referred to herein as the sequence
of the macromolecule.

[0032] A "ligand" is a molecule that is recognized by a particular
receptor. Examples of ligands that can be investigated by this invention
include, but are not restricted to, agonists and antagonists for cell
membrane receptors, toxins and venoms, viral epitopes, hormones, hormone
receptors, peptides, enzymes, enzyme substrates, cofactors, drugs (e.g.
opiates, steroids, etc.), lectins, sugars, polynucleotides, nucleic
acids, oligosaccharides, proteins, and monoclonal antibodies.

[0033] A "receptor" is molecule that has an affinity for a given ligand.
Receptors may-be naturally-occurring or manmade molecules. Also, they can
be employed in their unaltered state or as aggregates with other species.
Receptors may be attached, covalently or noncovalently, to a binding
member, either directly or via a specific binding substance. Examples of
receptors which can be employed by this invention include, but are not
restricted to, antibodies, cell membrane receptors, monoclonal antibodies
and antisera reactive with specific antigenic determinants (such as on
viruses, cells or other materials), drugs, polynucleotides, nucleic
acids, peptides, cofactors, lectins, sugars, polysaccharides, cells,
cellular membranes, and organelles. Receptors are sometimes referred to
in the art as anti-ligands. As the term "receptors" is used herein, no
difference in meaning is intended. A "Ligand Receptor Pair" is formed
when two macromolecules have combined through molecular recognition to
form a complex. Other examples of receptors which can be investigated by
this invention include but are not restricted to:

[0034] a) Microorganism receptors: Determination of ligands which bind to
receptors, such as specific transport proteins or enzymes essential to
survival of microorganisms, is useful in developing a new class of
antibiotics. Of particular value would be antibiotics against
opportunistic fungi, protozoa, and those bacteria resistant to the
antibiotics in current use.

[0035] b) Enzymes: For instance, one type of receptor is the binding site
of enzymes such as the enzymes responsible for cleaving
neurotransmitters; determination of ligands which bind to certain
receptors to modulate the action of the enzymes which cleave the
different neurotransmitters is useful in the development of drugs which
can be used in the treatment of disorders of neurotransmission.

[0036] c) Antibodies (Abs): For instance, the invention may be useful in
investigating the ligand-binding site on the antibody molecule which
combines with the epitope of an antigen of interest; determining a
sequence that mimics an antigenic epitope may lead to the-development of
vaccines of which the immunogen is based on one or more of such sequences
or lead to the development of related diagnostic agents or compounds
useful in therapeutic treatments such as for auto-immune diseases (e.g.,
by blocking the binding of the "anti-self" antibodies). There are
monoclonal antibodies (mAb) and polyclonal antibodies (pAb).

[0037] d) Nucleic Acids: Sequences of nucleic acids may be synthesized to
establish DNA or RNA binding sequences. Certain sequence of nucleic
acids, called aptamer, can bind to proteins or peptides.

[0038] e) Catalytic Polypeptides: Polymers, preferably polypeptides, which
are capable of promoting a chemical reaction involving the conversion of
one or more reactants to one or more products. Such polypeptides
generally include a binding site specific for at least one reactant or
reaction intermediate and an active functionality proximate to the
binding site, which functionality is capable of chemically modifying the
bound reactant.

[0039] f) Hormone receptors: Examples of hormones receptors include, e.g.,
the receptors for insulin and growth hormone. Determination of the
ligands which bind with high affinity to a receptor is useful in the
development of, for example, an oral replacement of the daily injections
which diabetics take to relieve the symptoms of diabetes. Other examples
are the vasoconstrictive hormone receptors; determination of those
ligands which bind to a receptor may lead to the development of drugs to
control blood pressure.

[0040] g) Opiate receptors: Determination of ligands which bind to the
opiate receptors in the brain is useful in the development of
less-addictive replacements for morphine and related drugs.

[0041] A "linker" molecule refers to any of those molecules described
supra, such as for example molecular probes, and preferably should be
about 4 to about 100 atoms long to provide sufficient exposure. The
linker molecules may be, for example, aryl acetylene, alkane derivatives,
ethylene glycol oligomers containing 2-10 monomer units, diamines,
diacids, amino acids, among others, and combinations thereof.
Alternatively, the linkers may be the same molecule type as that being
synthesized (i.e., nascent polymers), such as polynucleotides,
oligopeptides, or oligosaccharides.

[0042] The term "fluid" used herein means an aggregate of matter that has
the tendency to assume the shape of its container, for example a liquid
or gas. Analytes in fluid form can include fluid suspensions and
solutions of solid particle analytes.

[0043] The term "attached," as in, for example, the "attachment" of a
molecular probe to a ferromagnetic microdisk, includes covalent binding,
adsorption, and physical immobilization. The terms "associated with,"
"binding," and "bound" are identical in meaning to the term "attached."
Attachment of molecular probe to ferromagnetic microdisk, or molecular
probe to bioanalyte, can be permanent or reversible.

[0044] The term "permalloy" refers to a nickel iron magnetic alloy.
Generically, it refers to an alloy with about 20% iron and 80% nickel
content (i.e., Ni80Fe20). Permalloy has a high magnetic permeability, low
coercivity, near zero magneto striction, and significant anisotropic
magnetoresistance. This alloy is used, for example, in transformer
laminations, and magnetic recording head sensors. Permalloy's electrical
resistivity generally varies within the range of 5% depending on the
strength of the magnetic field. The low magnetostriction is helpful for
industrial applications, where variable stresses in thin films would
otherwise cause a ruinously large variation in magnetic properties. Other
compositions of permalloy are available, designated by a numerical prefix
denoting the percentage of nickel in the alloy, for example 45 permalloy
containing 45% nickel, and 55% iron. Molybdenum permalloy is an alloy of
81% nickel, 17% iron and 2% molybdenum.

[0045] Other ferromagnetic materials are encompassed by embodiments of the
invention. For example, ferromagnetic microdisks can be fabricated with
substances such as CoNiFe, CoFe, CoFeCu, CoZrTa, and other ferromagnetic
metals or alloys.

[0046] Embodiments of the invention relate generally to ferromagnetic
microdisks bound to molecular probe, methods of detecting target
bioanalyte using ferromagnetic microdisks, and kits (such as for using in
the laboratory setting) containing the reagents necessary to make, and/or
use ferromagnetic microdisks for bioanalyte detection, depending on the
user's planned application. The methods and products allow the
fabrication of ferromagnetic microdisk/molecular probe complexes, and
their use in the detection of biological molecules (bioanalytes) with
high sensitivity, little or no signal decay, improved safety,
convenience, and lowered cost of use and disposal. The embodiments are
especially directed to utilizing ferromagnetic microdisks exhibiting
unique resonance frequency as "tags," and identifying the tags using
detection of the unique resonance frequency by known means, or other
detection methods wherein ferromagnetic microdisks can be detected and/or
observed. Ferromagnetic microdisks can be used in solution or attached to
a substrate for bioanalyte detection, depending on user needs.

[0047] One embodiment is a ferromagnetic microdisk, constructed from a
ferromagnetic material, that exhibits a unique resonance frequency
determined by the geometry of the microdisk, and a molecular probe
attached to the ferromagnetic microdisk. Preferably, the ferromagnetic
material is permalloy.

[0048] Preferably, ferromagnetic microdisks have a diameter of less than
about 3 μm and a thickness of less than about 50 nm. More preferably,
ferromagnetic microdisks have a diameter of about 1.1 μm to about 2.2
μm, and a thickness of about 20 nm to about 40 nm.

[0049] Preferably, ferromagnetic microdisks are fabricated, and the
geometry is determined, by photolithography.

[0050] In embodiments of the invention, the ferromagnetic microdisks
exhibit a unique resonance frequency ranging from about 25 mHz to about
400 mHz. Preferably, the unique resonance frequency is from about 80 to
about 272. mHz. More preferably, the resonance frequency is about 83,
162, or 272 mHz.

[0051] Preferably, ferromagnetic microdisks of the invention are attached
to molecular probes. Molecular probes include, for example, antibody,
antigen, ligand, receptor, aptamer, or a nucleic acid. More preferably,
the molecular probe comprises an antibody or a nucleic acid. More
preferably, the molecular probe attached to the ferromagnetic disk
comprises protein.

[0052] Another embodiment is a method of detecting a target bioanalyte,
such as a biomolecule of interest, with a ferromagnetic microdisk by
attaching ("bioconjugating") one or more molecular probes to one or more
ferromagnetic microdisks that are made of a ferromagnetic material and
exhibit a unique resonance frequency; contacting the bioconjugated
ferromagnetic microdisk with at least one target bioanalyte of interest;
binding the molecular probe to the target analyte, and detecting the
unique resonance frequency exhibited by the ferromagnetic microdisk,
thereby detecting presence of the ferromagnetic microdisk and thus the
presence of at least one target bioanalyte which has bound to the
molecular probe on the ferromagnetic microdisk.

[0053] Preferably, the molecular probe attached to the ferromagnetic disk
is chosen from an antibody, antigen, ligand, receptor, aptamer, and
nucleic acid. More preferably, the molecular probe comprises protein.

[0054] In one embodiment of the invention, the target bioanalyte is
present in a solid sample. In another embodiment, the target bioanalyte
is present in a solution. In certain embodiments, the target bioanalyte
is bound to a solid or semisolid support or matrix.

[0055] Preferably, the target bioanalyte is present in vitro. More
preferably, the target bioanalyte is present in vivo.

[0056] In other embodiments of the invention, the bioanalyte to be
detected is present in a non-living sample, such as for example, a food
sample, soil sample, or water sample. Embodiments of the invention are
not limited to biological samples or tissues, and can be used in
industry, geology, an contamination detection.

[0057] Another embodiment of the invention is a kit that includes one or
more ferromagnetic microdisks and one or more molecular probes attached
to the ferromagnetic microdisk, wherein the ferromagnetic microdisks
comprise a ferromagnetic material and exhibit a unique resonance
frequency determined by the geometry of the microdisk.

[0059] Preferably, the molecular probe is selected from the group
consisting of antibody, antigen, ligand, receptor, aptamer, and nucleic
acid. More preferably, the molecular probe comprises protein.

[0060] Embodiments of the invention encompass ferromagnetic microdisks
that include a ferromagnetic material and exhibit a unique resonance
frequency determined by the geometry of the microdisk. The manufacture
and resonance frequency characteristics of ferromagnetic microdisks have
been described in the art (see Novosad, et al., Magnetic Vortex Resonance
in Patterned Ferromagnetic Dots, Physical Review, B72, 024455-1-5 (2005),
the entire disclosure of which is hereby incorporated by reference).

[0061] Embodiments of this invention addresses the the problem of: (1)
target bioanalyte detection when present in low levels in the target
sample; (2) highly sensitive detection of antigens, antibodies, and
viruses, and other bioanalytes; (3) using markers or tags that are safe
to the use and environmentally sound, are easy to use, and that exhibit a
signal that does not decay or degrade over time or with use. As a result,
embodiments of the invention simplify sample preparation and
significantly lower the costs and biohazards associated with bioanalyte
detection.

[0062] As described, embodiments of this invention provide highly
sensitive devices and methods for bioanalyte detection. With a
biomolecular probe attached to ferromagnetic microdisk, mass sensitivity
can be estimated as follows. Note that each spectrum shown in FIG. 3 was
collected from ˜1200 microdisks. Mass of biomolecule is ˜1000
kDa (kilo Dalton. 1 Da ˜1.66×10-27 kg). As examples, the
mass of yeast protein and titins protein is 53 kDa and 3000 kDa,
respectively. The mass sensitivity can be calculated as follows.

1200 biomolecules×1000 kDa=1,200,000 kDa˜2×10-18
kg.

The mass sensitivity is therefore ˜10-18 kg.

[0063] In the embodiments of the invention, permalloy films are preferred
substrate for ferromagnetic microdisks. Such films have been shown to be
useful for the fabrication of ferromagnetic microdisks. However, other
ferromagnetic materials can be used, such as for example CoNiFe, CoFe,
CoFeCu, CoZrTa, and other ferromagnetic metals or alloys.

[0064] Some of the technical advantages of the embodiments of the
invention include the following: [0065] (1) Target bioanalytes (e.g.,
proteins or nucleic acids) need not be labeled or amplified (DNA). This
is because ferromagnetic microdisk detection is a label-free strategy
that is superior to fluorescence technology in which target samples must
be labeled with fluorescent molecules that are prone to photobleaching.
Thus, tedious, error-prone, costly, and environmentally unfriendly (e.g.,
radioisotope labeling) sample preparation steps can be avoided. [0066]
(2) Multiple targets can be detected at the same time in one sample by
using multiple ferromagnetic microdisks variable bioconjugated molecular
probes. Ferromagnetic microdisks can be "free" in a sample, or
alternatively, bound to a solid or semisolid substrate, such as for
example, an array, wherein a test sample is applied to the array and for
bioanalyte detection with complementary molecular probes. Ferromagnetic
microdisks can be used in discrete units, i.e., ferromagnetic microdisks
with one type of molecular probe that is specific to one particular
bioanalyte, or alternatively, ferromagnetic microdisks can be used with a
library of molecular probes that are specific to a myriad of bioanalytes.
This latter embodiment is advantageous in the fabrication and use of
ferromagnetic microdisks in an array. [0067] (3) Each bioanalyte molecule
detection can be verified by duplication using the same detection probe
but with a different ferromagnetic microdisk. The multiplicity of
ferromagnetic microdisk detection allows redundant measurement, thereby
providing greater control increased credibility of the test results.
[0068] (4) The structure of ferromagnetic microdisks and their respective
attached biomolecular probes can be easily fine-tuned to meet specific
applications in diagnostics and drug discovery.

[0069] Embodiments of this invention have several useful applications. For
example, ferromagnetic microdisks can be employed for the ultra-sensitive
detection of bioanalytes including, antibodies, antigens, biomarkers,
allergens, ligands, metabolites, virus, bacteria, tumor cells, etc. The
ability to detect, locate, and/or quantify bioanalytes allows for
diagnostic use, treatment, and/or monitoring of specific diseases,
physiological conditions (normal or abnormal), conditions, and therapies.
For example, detection of abnormal proteins in human disease could
detected. As another example, the normal signal transduction inside, or
outside cells could be detected and monitored. It is envisioned that
embodiments of the invention could be used in vivo or in vitro for
screening purposes, i.e., high throughput methods of evaluating
pathological conditions. High-throughput drug discovery screening is
another example where embodiments of the invention would be useful.

[0070] Resonance frequency detection, for example with equipment to
measure resonance frequency such as a network analyzer could be employed
in both normal physiological systems (e.g., at the cellular, tissue, and
whole animal level), and also in pathological states for disease
evaluation. Embodiments of the invention are also useful in flow
cytometry, environmental monitoring, and food analysis.

[0071] In order to provide users with the ability to efficiently utilize
embodiments of the invention, the present invention contemplates methods
and kits for screening samples containing suspected analytes of interest
that could be detected with ferromagnetic microdisks. The kits contain
the reagents necessary to manufacture ferromagnetic microdisks, including
the disks, reagents for attaching one or more molecular probe(s) that can
bind to the target bioanalyte of interest. Such kits are advantageous for
users who want to create ferromagnetic microdisks and attach molecular
probes that will be useful in specific applications, such as for example,
locating, quantifying, and or analyzing particular target bioanalytes of
interest.

[0072] For example, one kit contains all the reagents necessary for the
production of ferromagnetic microdisks, and a molecular probe, such as a
particular receptor, that is conjugated to the ferromagnetic microdisks;
in this manner, the ferromagnetic microdisk has been "tagged" with a
molecular probe. The particular receptor, when contacted to a sample of
interest, will bind to a cellular protein (bioanalyte) of interest that
is specific for, or complementary to, the molecular probe attached to the
ferromagnetic microdisk. The target sample containing bioanalyte can be
derived from, for example, a cell culture (i.e., in vitro), or from a
mammalian sample (i.e., in vivo). After contacting the tagged
ferromagnetic microdisk with bioanalyte of interest, the ferromagnetic
microdisk is detected based on its unique resonance frequency (using, for
example, detectors that recognize the characteristic magnetic vortex
resonance frequency emitted by the ferromagnetic microdisk), thereby
detecting the presence (or absence), quantity, and location of the target
cellular protein (i.e., bioanalyte) of interest. This example is merely
illustrative, and not intended to be limiting.

[0073] Although embodiments have been described in which small molecules
and proteins are described as being the analytes, it is understood,
however, that the same process and tools can be used to detect the
binding of a variety of analytes to one another and the invention is not
limited to just the binding of small molecules to proteins.

EXAMPLE 1

[0074] FIG. 1 shows an exemplary ferromagnetic microdisk that is attached
to a molecular probe ("linker") which acts as a binding partner to a
bioanalyte of interest. The ferromagnetic microdisk is made of a
ferromagnetic material, such as permalloy, that is well known in the art.
Ferromagnetic microdisks exhibit a unique resonance frequency based on
their geometry, such as the diameter and thickness of the microdisk; by
altering the geometric parameters, ferromagnetic microdisk are made with
specific unique resonance frequencies.

[0075] As shown in FIG. 1, tagged biomolecule(s) approach the read line
(which are simply conducting lines such as copper), the ferromagnetic
microdisk is detected by evaluating the characteristic magnetic vortex
resonance frequency. Read lines may be microwave coplanar waveguides used
to generate magnetic field and collect (i.e., detect) the frequency
spectrum. Microchannel may be formed in order to provide closed volume
within which all biomolecules and ferromagnetic microdisks can be
confined.

[0076] The ferromagnetic microdisk, preferably the outer surface thereof,
is bioconjugated to a molecular probe and the complex is used for
bioanalyte detection. The ferromagnetic microdisks are functionalized
with an molecular probe, such as an amine group. Various bioanalytes of
interest in a sample are contacted with, and conjugated to, the
functionalized ferromagnetic microdisks through bioconjugation methods
for that are well known in the art, such as hybridization. Bioanalytes of
interest to be detected include, for example, proteins, antibodies,
enzymes, nucleic acids (DNA, RNA, oligonucleotides), antigen, peptides,
ligands, receptors, small molecules, metabolites, etc. Although the
biological applications of the bioconjugated ferromagnetic microdisk is
are immense, detection of signature antibody, autoantibody, antigen,
virus and bacterium are of special interest for disease diagnostics and
treatment monitoring.

[0077] The bioanalyte of interest is now bound to one or more
ferromagnetic microdisks because the molecular probe (conjugated to the
surface of the ferromagnetic microdisk) also binds to the bioanalyte of
interest. The bioanalyte of interest is now located and quantified by
detection of the unique resonance frequency emitted by the bound
ferromagnetic microdisk. The resonance frequency is detected with a
magnetic signal detector and network analyzer; such methods that are well
known in the art for detecting and quantify magnetic signals.

EXAMPLE 2

[0078] FIG. 2 shows a method of manufacturing ferromagnetic microdisks.
Ferromagnetic microdisks are made by photolithography methods well known
in the art, wherein ferromagnetic materials such as permalloy undergo
spin-coating, resist development, and E-beam metallization. The unique
resonance frequency is determined, and can be altered by, changing the
geometry of the microdisk, including changing the diameter and thickness
of the microdisk. The manufacture and resonance frequency characteristics
of ferromagnetic microdisks have been described in the art (see Novosad,
et al., Magnetic Vortex Resonance in Patterned Ferromagnetic Dots,
Physical Review, B72, 024455-1-5 (2005); see also Novosad, et al.,
Ferromagnetic Microdisks: Novel magnetic Particles for Biomedical
Applications, NSTI-Nanotech, vol. 1:308-311 (2005) (the entire
disclosures of which are hereby incorporated by reference).

EXAMPLE 3

[0079] Permalloy ferromagnetic microdisks are manufactured in a variety of
thicknesses and diameters by methods known in the art. Three microdisk
geometries are made to demonstrate the effect of variable geometry on
resonance frequency:

[0080] FIG. 3 shows the data for these microdisks. The resonances at 83,
162, and 172 mHz, respectively, are in agreement with the
eigenfrequencies of the collective spin excitations simulated
micromagnetically and analytically. The number of frequency sweeps
average 320, 160, and 640 respectively.

[0081] The resonance frequency of each ferromagnetic microdisk can be
detected, thereby demonstrating the presence of one or more microdisks
having that unique resonance frequency. In this manner, a user can
bioconjugate a molecular probe to a ferromagnetic microdisk of known
unique resonance frequency. The molecular probe/ferromagnetic disk is a
"complex" that can be used to target, locate, identify, and quantify
bioanalytes of interest that are complementary, or bind to, the molecular
probe. Molecular probes, methods of bioconjugation (molecular probe to
ferromagnetic disk), as well as binding of ferromagnetic disk/molecular
probe complex to bioanalyte of interest (for example, hybridization or
chemical linking) are well known in the art and easily within the skill
of a person in the art.

[0082] Conveniently, either the sample or the ferromagnetic disk/molecular
probe complex(es) can be applied and bound to a solid or semi solid
substrate, such as for example, glass. In this manner, manual or
automated dispensers (such as for example, robots) can be used to produce
"arrays" containing multiple different ferromagnetic disks, each with
their own unique resonance frequency or molecular probe. Accordingly, a
sample can be contact to the ferromagnetic disk array, and several
different bioanalytes of interest can be evaluated in one experiment,
allowing for high throughput uses. Array formats and technologies, such
as robotic dispensers, array substrates, and pattern algorithms are well
known in the art. Advantageously, in such array or high throughput uses,
the unique resonance frequency "signal" emitted by the ferromagnetic
disk/molecular probe complex does not fade or decay over time. Also, such
signals are not harmful to users, as are radioisotopes. Ferromagnetic
disks can be discarded without concern for radioactive contamination of
the environment.

[0084] This application discloses several numerical range limitations that
support any range within the disclosed numerical ranges even though a
precise range limitation is not stated verbatim in the specification
because the embodiments of the invention could be practiced throughout
the disclosed numerical ranges. Finally, the entire disclosure of the
patents and publications referred in this application, if any, are hereby
incorporated herein in entirety by reference.